Abstract

Hypoxia is an important phenomenon in many physiological processes and involved in many human diseases including cancer. Inflammation can lead to significant hypoxia in tissues. The study of hypoxia has been complicated with the lack of proper instrumentation to induce hypoxia in cells and image cells under hypoxic conditions. Here, we describe a live cell-based method to conveniently measure hypoxia using a new Image-iT® Hypoxia Probe and a specialized microscope incubator which can control oxygen concentrations down to 1%, The Image-iT® Hypoxia Probe is an oxygen sensing fluorescent probe, is quenched with increasing oxygen concentrations, and has excitation and emission peaks of 483 and 616 nm respectively. The probe is sensitive to varying concentrations of oxygen and can detect as low as 1% O2 concentrations in cells. Imaging of cells with the Image-iT® Hypoxia Probe in the incubator prevents re-oxygenation of cells and gives more precise measurement of hypoxia in cells, allowing for reversible and dynamic measurements of hypoxia in cells. Using this system, we measured hypoxia in several cell lines including A549, HeLa and U-2 OS. The Image-iT® Hypoxia Probe is also very good at detecting hypoxia in 3D tumor spheroids generated using different methods. The new hypoxia probe gives good signal to noise with more than 3-fold changes at 5% O2 levels with robust statistics. The Image-iT® Hypoxia Probe provides a good system for precise, robust and reproducible measurements of hypoxia in cells.